Overexpression of thymidylate synthase (TS) is observed in a wide range of human cancers where it is associated with advanced disease and resistance to commercially available TS inhibitors. My Lab demonstrated that activated expression of TS, an essential enzyme for DNA synthesis and repair, plays a direct role in promoting tumorigenesis. This was an unexpected observation as elevated TS levels were previously believed to reflect a passive biomarker for tumor cell growth. Our observation changed the paradigm of TS as a biomarker and has great significance for cancer treatment as it refocuses attention on the importance of TS as a tumor-promoting signal. While our published data proposed that TS plays an active role in promoting tumorigenesis, questions remain whether elevated TS cooperates with other common genetic lesions to enhance the neoplastic process. To address this question we developed a new genetically engineered mouse model (GEMM) with Ink4a/Arf-/- mutation and overexpression of human TS (designated hTS/Ink4a/Arf-/-). We showed for the first time that TS activation in Ink4a/Arf null mice is associated with accelerated tumor growth, larger tumor size and more advanced stage of the disease. We also showed that activated TS promote the growth and metastasis of previously established tumors rather than inducing development of a new tumor spectrum. Ink4a/Arf is one of the most commonly mutated loci in human cancer and its products encode tumor suppressor proteins required for G1 arrest. The objectives of this proposal are: i) to determine how TS overexpression promotes tumorigenicity in hTS/Ink4a/Arf-/-mice. We hypothesize that TS overexpression drives DNA replication stress by deregulating replication factors and nucleotide pool ratios that result in double strands break and genomic instability in the absence of INK4a/ARF checkpoints. ii) to develop new potent TS allosteric inhibitor analogues. We have discovered a new strategy that takes advantage of high TS subunit cooperativity to identify small molecule inhibitors of TS. Targeting TS dimer interface with allosteric inhibitors overstabilize the dimer structure resulting in loss of subunit cooperativity and inhibition of TS enzymatic function. We will identify potent derivatives to prevent or reverse drug resistance that limits the effectiveness of current therapy. Our new TS allosteric inhibitors do not cause TS overexpression thus these new small molecules may overcome the problem of TS overexpression associated resistance and iii) to test anti-tumor activity of allosteric inhibitors alone and in combination with conventional TS inhibitors in our new GEMM with activated hTS. In summary, this research project proposes a new approach to control TS mediated tumorigenesis with the goal to reduce drug resistance and optimize TS inhibition in cancer treatment. Determination of how TS overexpression promotes tumorigenicity and the development of novel TS inhibitors will provide a new focus toward targeting TS for therapeutic intervention and for cancer treatment.